Methods and systems for monitoring multiple optical signals from a single source

ABSTRACT

Methods and systems for monitoring a plurality of different optical signals from a single source of such signals, where each such different optical signal is spatially separated from other such signals and directed to different detectors or locations upon a single detector, which direction is generally accomplished through the use of a small number of optical components and/or manipulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/201,768 filed on Aug. 11, 2005 which is incorporated herein byreference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

The individual identification, distinction and/or quantitation ofdifferent optical signals from a collection of such signals is of majorimportance in a number of different fields. Of particular note is theuse of multiplexed analytical operations, e.g., chemical assays, etc.,which employ optical signaling events that have different opticalcharacteristics which may then be identified and potentially quantifiedseparately from each other optical signal. Such analytical assaysinclude medical diagnostic tests, food and other industrial processanalyses, and basic tools of biological research and development. Whilea wide variety of optical and chemical approaches have been appliedtoward analysis of these signals, such systems often include a level ofcomplexity and/or cost that detracts from the overall utility of theapproach, particularly for operations that require high levels ofsensitivity. The present invention addresses these shortcomings of othersystems and methods.

BRIEF SUMMARY OF THE INVENTION

The present invention generally provides methods and systems fordetecting and monitoring a plurality of different optical signals from asingle, preferably confined source of such signals. In preferredaspects, such systems and methods are applied to the detection ofluminescent or fluorescent signals from fluid borne materials andparticularly reactants and/or products of chemical, biochemical orbiological reactions of interest.

In a first aspect, the present invention provides methods of detectingoptical signals, where such methods comprise providing a source of atleast first and second optical signals wherein the first optical signalcomprises an optical characteristic different from an opticalcharacteristic of the at least second optical signal. In preferredaspects, the optical characteristic is a wavelength of the opticalsignals. The optical signals are directed to different locations on adetector, e.g., by passing the signals through an optical train thattransmits the first and second optical signals in divergent paths, andthen received at different locations on one optical detector.

In a related aspect, the method of detecting optical signals, comprisesproviding a source of a plurality of different optical signals, whereineach different optical signal comprises a wavelength different from eachother optical signal, and spatially separating the plurality ofdifferent optical signals and directing them to discrete locations onone optical detector.

In a further aspect, a method is provided for detecting optical signals,which method comprises providing a confined source of at least first andsecond optical signals wherein the first optical signal comprises adifferent optical characteristic, i.e., wavelength, from that of the atleast second optical signal. The signals are then spatially separatedand directed to first and second different locations on a first opticaldetector.

The present invention also provides for systems useful in carrying outthe foregoing methods. For example in one aspect, the invention providesanalytical systems, comprising a confined reaction region for containinga reaction mixture that produces at least first and second opticalsignals wherein the first optical signal comprises an opticalcharacteristic different from that of the at least second opticalsignal. Such systems also comprise an optical train in opticalcommunication with the confined reaction region, for receiving the firstand second optical signals and spatially separating the first and secondoptical signals and directing them to different locations on an opticaldetector.

Related systems of the invention comprise a confined reaction region forcontaining a reaction mixture that produces at least first and secondoptical signals wherein the first optical signal comprises a wavelengthdifferent from a wavelength of the at least second optical signal, anoptical train in optical communication with the confined reactionregion, for receiving the first and second optical signals and spatiallyseparating the first and second optical signals and directing them todifferent locations on an optical detector. In alternate aspects, theoptical train comprises a replaceable modular optical component thatspatially separates the first and second optical signals passingtherethrough. By selecting different modules from a collection orlibrary of modules, one can increase the usefulness of the overallsystem.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a simplified schematic illustration of the methods andsystem of the invention.

FIG. 2 provides a schematic illustration of the operation of the systemsand methods of the invention in monitoring multiple different opticalsignals over time.

FIG. 3 schematically illustrates one exemplary system according to thepresent invention in greater detail.

FIG. 4 schematically illustrates an alternate system configuration formonitoring multiple optical signals that differ in their relativepolarization, as opposed to other characteristics of light, e.g.,wavelength.

FIG. 5A shows different optical signals incident upon differentlocations of a single CCD camera chip, which were derived from a single,combined source, and subjected to the methods of the invention. FIG. 5Bshows the relative distance of separation between separated signals.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention is generally directed to devices, systems andmethods for the facile, efficient and cost effective analysis and/ormanagement of collections of optical signals and the data derived fromthose signals. Of particular interest is the application of thesedevices, systems and methods in analyzing reactions of interest, e.g.,chemical and biochemical reactions such as nucleic acid synthesis, andthe characterization of the steps involved in those reactions.

In general, the present invention is directed to methods, systems anddevices for measuring two or more different optical signals from asource of optical signals, by separating the optical signals from eachother and directing them to different detection functionalities, ordifferent locations, on a single optical detector. By separatelydetecting the different optical signals one can recognize the occurrenceof the causal events for each signal. In addition, by doing so withinfew detectors or a single detector or detector array, one can reduce thecomplexity and cost of systems and their associated control and analysisprocesses, while concurrently increasing their efficiency and/orsensitivity.

While the overall systems and methods of the invention may be employedbroadly in a wide range of different applications, of particularinterest is the use of these systems and methods in the analysis andcharacterization of chemical and/or biochemical reactions, which eithernaturally or artificially produce such differing optical signals duringthe reaction process. There are a wide variety of different analyticalreactions that produce multiple optical signals that would benefit fromthe present invention. These include reactions that use optical signalsof differing wavelengths, e.g., fluorescent and/or fluorogenic reactantsor products, luminescent reactants or products, chromophoric and/orchromogenic reactants or products, etc., and reactions that use opticalsignals that differ in other characteristics, e.g., shifts inpolarization or phase modulation of emitted light. In general, as usedherein, reference to a wavelength of an optical signal includes awavelength range for that signal. In particular, optical signals, e.g.,emitted fluorescence, luminescence, or the like, will span a portion ofthe optical spectrum which portion may span a range of from 1 nm to 30nm up to 100 nm or more within the overall spectrum. In terms of thepresent invention, optical signals of different wavelengths denotesignals whose wavelength range is distinguishable from the other. Thuswhile little or no overlap of the wavelength ranges for differentsignals would be ideal, a substantial amount of wavelength overlap maybe tolerated, provided that signals may be individually identified.Methods of identification and distinction of signals from signal overlapor noise in optical systems, i.e., through the use of optical componentsand/or through stringent data selection, is well known in the art. In aparticularly preferred aspect, the analytical methods and systems of theinvention are applied in nucleic acid analyses and particularly nucleicacid sequence analyses.

Because the methods and systems of the invention have reducedcomplexity, and as a result, higher sensitivity, they are particularlyuseful in applications where the optical signals to be detected arerelatively weak, e.g., low light levels, few signal events, etc. Inparticular, because the systems employed in the invention minimize thenumber of optical manipulations that signals are put through, theoverall efficiency losses of the system that are summed from each suchmanipulation are likewise reduced. For example, where optical signalsare passed through multiple beam splitting, refocusing, filtering, etc.operations, losses associated with each stage can dramatically reducethe sensitivity of the overall assay. Additionally, losses associatedwith examining only a separate portion of the optical spectrum of theoverall signal, e.g., using restrictive band-pass filters and the like,can further reduce the amount of signal that could otherwise be used inthe detection operation. In the case of the methods and systems of theinvention, the entire spectrum of the overall signal is subjected todetection, and selection of each different signal component is a matterof selecting the location on a single detector, e.g., which pixels in adetector array, should be applied toward assessing a given signal,rather than cutting off a portion of signal before it is ever detectedthrough, e.g., optical cut-off filtering.

While many applications begin with more than adequate signal strength toallow for such losses, some applications operate at signal levels that,when combined with the efficiency losses, are either below the level ofmeaningful detection of the overall system, or the effect of interest isa change to the optical signal where such change is within the noiselevel of the system, e.g., the signal is so small as to beindistinguishable from random fluctuations in signal intensity. Examplesof these low signal types of applications include, for example, lowconcentration chemical analyses such as single or few moleculereactions, and the like, where very few or even a single detectablemolecule may be all that is available to be detected at any given time.

II. Methods

As noted above, in one aspect, the invention is directed to methods ofdetecting optical signals, from a source of a plurality of differentoptical signals, by separating the different optical signals from eachother and directing at least a portion of them to discrete locations onone optical detector or detector array. In the case where multiplesignals are detected at different locations on a single detector, itwill be understood that such detector includes or is capable of beingconfigured to provide signal information for signals incident thereon,that correlate not only the signal intensity and time, but also theposition or location upon the array at which such signal is incident.Simple examples of such detectors include array type detectors as aregenerally known in the optics art, and certain examples of which aredescribed in greater detail herein. In the case where single pointsignals are to be detected at discrete detectors, it will be understoodthat position information of an incident signal is provided by thelocation of each individual detector (typically although not necessarilyof a plurality of individual detectors), rather than a location withinone single detector or detector array.

While the methods of the invention could be applied to a wide variety oftypes of sources of optical signals, in preferred aspects, the source ofoptical signals comprises a confined source. The confined sources of theinventions are typically characterized in that one or more components ofthe source that produce the particular optical signals are confined inspace, and are not flowing into and or out of the confined source duringthe detection. Such confined sources are in contrast to systems wheresignal producing components, reactants, or the like are actively flowingpast a point of detection in a conduit. Notwithstanding the foregoing,components of the signal producing mechanism employed in the inventionmay be diffusing into and out of the confined space, while still fallingwithin the parameters set forth herein. In many cases, however, one ormore components that contribute to the signaling mechanism will beimmobilized within the confined space.

The confined nature of the sources is of particular value where theoptical signals result from reactive chemical species and particularlyfluid borne reactive chemical species, e.g., aqueous and/or organicfluids. In particular, in the case of fluid sources of differing opticalsignals, the confined nature of the source would not permit the movementof such fluids into or out of the confinement during detection. Examplesof fluid confinements include, e.g., conventional multiwell analysisplates, e.g., 96, 384 or 1536 well plates. Other examples ofconfinements for such fluid reactants include nanoscale wells orapertures, i.e., zero mode waveguide structures as described inPublished U.S. Patent Application No. 2003/0174992 A1, which isincorporated herein by reference in its entirety for all purposes, whichserve as both physical confinements and optical confinements, e.g.,limiting the amount of light that penetrates into the waveguide and thuseffectively limiting the volume from which signals, e.g., fluorescentsignals, emanate. Such zero mode waveguides are particularly useful inthe exploitation of the invention, in that they provide the ability tomonitor different optical signals from vary small volumes, e.g., fluidborne reactants, allowing monitoring of interactions between fewmolecules, etc. Thus, while a zero-mode waveguide may represent theconfined space, the observed volume of that confined space is a fractionof the volume of such space, as is determined in part by the dimensionsof the waveguide. This fractional observed volume represents a furtherconfinement of the signal source. Of particular interest is the use ofsuch confined volumes in single molecule interactions, such as DNAsequence identification through the stepwise reaction of labelednucleotide analogs with a nucleic acid polymerase in template dependentnucleic acid synthesis, molecular interaction monitoring, i.e., DNAhybridization, immunoassays, enzymatic reactions, and the like.

In addition to structural confinement, e.g., using wells, reservoirs orthe like, confinement may additionally or alternatively comprisechemical immobilization of chemical species that produce one or more ofthe optical signals, i.e., either in place of or in addition to anystructural confinement. Examples of such chemical confinement includecovalent, van der waals or other associative interactions betweenchemical species and substrate surfaces, use of chemical interactions tocreate structural confinements, e.g., substrates having hydrophilicregions surrounded by hydrophobic barriers to confine fluid and chemicalspecies, and the like. In the case where confinement denotes chemicalimmobilization of reactants in a given location, a variety of differentimmobilization techniques may be employed, including, e.g., covalentlinkage of reactants onto surfaces of supports or substrates, includingfor example silane or epoxide linkages. Likewise, other associativelinkages may be employed using, e.g., complementary binding pairs tocouple reactants to substrates or supports. Such linkages include, e.g.,antibody/antigen linkages, biotin/avidin linkages, and the like. In thecase of chemically created structural confinements, again, a variety oftechniques are available for providing such ‘structures’ on substrates.In particular, hydrophobic barriers may be created by providingalkylsilane groups on otherwise hydrophilic silica surfaces. Suchmaterials are readily patterned onto substrate surfaces usingconventional photolithographic techniques, screen printing, ink-jetprinting or the like, to define hydrophilic confines surrounded byhydrophobic barrier regions.

As alluded to above, in preferred aspects the optical signals emanatingfrom the source derive from reactive chemical species, where thereaction of such species either produces, extinguishes, increases,decreases, or otherwise alters the characteristic of the opticalsignals. Such reactive species include chromogenic or chromophoricreactants, e.g., that produce a shift in the transmissivity of thematerial to light of one or more wavelengths, i.e., changing color uponreaction. Reactant species that emit light, either with the use of anactivating light source (fluorescent or fluorogenic) or without such anexcitation source (luminescent) are preferred for use in the methods ofthe invention. Further, in the context of the invention, such reactivespecies are most preferably contained in fluid solutions and areprovided as reaction mixtures where the different optical signals resultfrom the substrates, the products, or combinations of the two.

In preferred aspects, as noted above, the different optical signals tobe detected are comprised of light of differing wavelengths, e.g.,emitted by different fluorophores where such emissions have differentwavelength spectra, or transmitted by different chromophores where suchtransmissions are at different wavelength spectra. In such cases, thetwo or more different optical signals are spatially separated, e.g.,through the use of a beam splitter in combination with one or moredichroic filters, or through the use of a prism or optical grating, andthe different signals are directed to different locations on an opticaldetector or detector array. In alternate aspects, the different opticalsignals may differ in other characteristics, such as their relativepolarity, their modulation phase or frequency, or the like, providedthat they may be spatially separated and directed to different regionson a detector or detector array, e.g., through the use of polarizing ordemodulation filters. Examples of biochemical assays based upon suchdiffering characteristics are described in, e.g., U.S. Pat. No.6,699,655, which discloses monitoring reaction progress by detecting ofthe relative polarity of fluorescent reactants and products (typicallyin combination with a polarization affecting agent) when excited withpolarized light.

The methods of spatial separation and/or direction of different opticalsignals to different locations on an optical detector or detector arrayis generally dependent upon the characteristic(s) of the differentoptical signals that is/are to be the basis of differential detection.For example, where the different optical signals differ in theirwavelength, separation and direction can be accomplished through the useof optical filters and/or prisms that selectively transmit or redirectlight of differing wavelengths in different manners and/or to differentdegrees. For example, a collected signal that comprises two differentwavelengths of light emanating from a confined source may be split intotwo beams, e.g., through the use of a dichroic filter to remove theother signal component, then passed through a barrier filter, therebyallowing only a portion of the overall signal to be directed to theoptical detector or detector array. In accordance with the invention,however, a simpler optical train is employed to separate optical signalsand direct them to different locations on a detector or detector array,or in some cases, to multiple different detectors or detector arrays. Inparticular, a wedge prism or optical grating may be employed to achievethis result. The use of such prisms or diffraction gratings providessimplicity to the optical train of the overall system and results in amore transmissive light path as compared to more complex opticalsystems. Additionally, in contrast to the use of cut-off filters, e.g.,dichroics, the entire spectrum of signal, or at least a more selectivelyfiltered portion of the signal, less, e.g., the reflective losses of theprism, may be directed to the detector or detector array. As a result,there is a greater amount of signal available for detection,manipulation and deconvolution. The simplicity of the invention providesfurther advantages in the flexibility of the system, where a singleinstrument may be easily configured to perform a wide range of differentoperations, e.g., perform operations that each employ different rangesof optical signals, by simply replacing an interchangeable prism portionof the optical train with another prism from a library or collection ofdifferent prisms. Reconfiguration of conventional multifilter opticaltrains, by contrast, would require much more substantial alteration,e.g., changing multiple filters, etc. In particular, in accordance withcertain aspects of the invention, the component of the optical trainthat spatially separates the optical signals may comprise a modular, andeasily replaceable component, such as a prism, multiple prisms, and/oroptical grating(s), that can be inserted into and ejected from anappropriate receiver slot on an instrument. Further, a given instrumentmay be supplied with ort suppliable with a library of such modularcomponents, where each of the components provides different opticaldispersion profiles for different optical signals or collections ofoptical signals, allowing facile reconfiguration of the separationcomponent by the end user and maximal usefulness and flexibility to theuser. Some exemplary optical trains are described in greater detailherein.

In keeping with the simplicity of the optical trains described herein,the ultimate detection of multiple optical signals in parallel istypically accomplished through the use of smaller numbers of detectors.In particular, detection of n optical signals (where n>1) is typicallyaccomplished through the use of at most, n−1 discrete detectors. Inparticularly preferred aspects, as many as 2, 3, 4, 5, 6 or moredifferent optical signals are directed to different locations on 1, orin cases of 3 or more signals, 2 or more discrete optical detectors ordetector arrays. In accordance with the invention, it will beappreciated that in cases where more than one signal is directed to morethan one location on a given detector, such detectors are not singlepoint detectors, e.g., simple photodiodes, but instead have a detectionarea that generates a signal that is indicative of the incidence of anoptical signal on the detector, as well as an indication of the locationon the detector where such signal was incident. Examples of suchdetectors include imaging detectors, such as charge coupled devices(CCDs), where each pixel element on the CCD constitutes a single pointdetector, but the overall device constitutes an array of detectors,where the detector signal indicates the pixel at which the signal wasincident and the intensity of that signal at that pixel. Similarly,larger diode array detectors may be used that include larger numbers ofphotodiodes spatially arranged and interfaced to provide both signalintensity and signal location information within the array.Notwithstanding the foregoing, simple point detectors may be used inconjunction with such detector arrays in accordance with the invention,e.g., where single signals are directed to a single detector, anddifferent signals are directed to different, or discrete detectors,rather than to regions on the same detector.

Although primarily and preferably directed at methods and systems wheremultiple optical signals are directed at one detector or detector array,or detectors that number less than the number of different opticalsignals to be detected, in certain alternative aspects, where opticalsignals that differ in wavelength are spatially separated using, e.g.,an optical grating or color dispersive prism, e.g., a wedge prism, eachdifferent signal is optionally directed to a different detector element,e.g., a point detector. In such cases, the incorporation of simple andcost effective separation optics, e.g., a prism or optical grating,provides enhanced efficiency over more complex optical trains, both interms of financial costs and in terms of optical efficiency. Thus, whilethe simplicity of using a single detector or detector array is notfound, efficiencies of costs may still exist where multiple lower costpoint detectors or lower resolution detector arrays are employed as thedetector elements. Further, such systems still retain the substantialefficiencies of cost over more complex systems and methods.

Based upon the spatial separation and direction, the incidence of anoptical signal at a particular location on the detector or detectorarray indicates that one of the two optical signals is being emitted ortransmitted from the confined source. If two or more locations on thedetector or elements on the detector array indicate the incidence of anoptical signal, it is indicative that two or more different opticalsignals are being emitted. By monitoring the particular location orelement that is indicating an incident signal, one can identify whichsignal is being emitted, and based upon the reaction being carried out,identify the reaction condition that is occurring, e.g., the generationof a given product or consumption of a given reactant.

A simplified schematic of the methods of the invention is illustrated inFIG. 1A. As shown, in a system 100, at least two different opticalsignals 102 and 104 emanate from a confined source 106 of such signals.As noted elsewhere herein, such confined sources may preferably bedefined locations that comprise fluid borne chemical reactants, such asreaction wells or regions, zero mode waveguides, etc. The differentoptical signals are then spatially separated (as shown by the divergentpaths of solid arrows 102 and dashed arrows 104) by passing thosesignals through an appropriate optical component, e.g., prism 108, anoptical grating or the like. Once separated, the signals are focusedthrough lens 110, e.g., an imaging lens, causing them to impinge ondetector array 112 at two different locations 114 and 116 on thatdetector array 112. The separation of signals is illustratedschematically in FIG. 1B. In particular, the combined optical signalsenter prism 108 as a signal as represented by spot 150. Once the signalshave passed through the spatial separation component of the opticaltrain, e.g., prism 108, and are focused onto the detector, they arespatially separated into their respective different optical signalcomponents, as represented by spots 152 and 154.

FIG. 2 schematically illustrates the detection operations over a periodof time, where the signals are concurrent or not. In particular, asshown, the system 100 is further connected to a recording/readoutsystem, schematically illustrated as plot 202. Over time, as indicatedby the horizontal axis of plot 202, different optical signals emanatefrom the confined source 106, either at different times (as shown attimes 204 and 206) or concurrently (at time 208). The optical signalsare detected on different locations of the detector 112, where eachlocation is separately connected to the recording system (e.g., andconnections 210 and 212). As a result, optical signals from a singleconfined source are separately detected and recorded, and can beattributed to a given point in time.

One exemplary use of the methods of the present invention is in theperformance of nucleic acid sequence analysis processes, andparticularly single molecule based processes that analyze nucleic acidsequences by monitoring the template dependent synthesis ofcomplementary nucleic acid sequences through the detection ofdifferently labeled nucleotide analogs that are incorporated into thegrowing synthesized strand. See, e.g., U.S. Patent Application Nos.2003/0044781A1, which is incorporated herein by reference in itsentirety for all purposes.

In one such method, a DNA polymerase enzyme is associated or complexedwith a template nucleic acid sequence, which is immobilized on thesurface of a substrate, attached through either the template or thepolymerase. The complex is exposed to appropriate polymerizationreaction conditions, including differently labeled nucleosidepolyphosphates, e.g., nucleoside triphosphates (NTPs), nucleosidetetraphosphates, nucleoside pentaphosphates, etc., or analogs of any ofthese, or other nucleoside or nucleotide molecules, that areincorporated by polymerase enzymes (all of which are referred to hereinas NTPs, for convenience), where each different NTP (e.g., A, T, G, orC) is labeled with fluorescent label having a different emissionwavelength profile. Incorporation of each different type of NTP producesa different optical signal indicative of the incorporation event. Forexample, in methods employing a confined volume containing theimmobilized polymerase/template complex, the incorporation of a givenfluorescent base results in that base being held within the detectionregion for longer periods than bases that are not incorporated. Bydetecting the signal associated with an incorporated base, one canidentify, in sequence, the bases that are incorporated in the templatedependent synthesis. In accordance with the invention, eachincorporation signal, generally characterized as a fluorescent pulse, isdirected to a different location on an optical detector array, andidentified based upon that location upon the detector array. Thus, asshown in FIG. 2, different optical signals are generated within a singleconfined source, although they may be generated at different times,e.g., sequentially as each base is incorporated.

In such cases, the polymerization reaction environment is confined byvirtue of its immobilization on the surface of the substrate, but isalso typically further, structurally confined, e.g., in a zero modewaveguide and/or within a reaction well in a multiwell plate.

In another example, a nucleic acid strand, e.g., a polynucleotide, isimmobilized upon the surface of a substrate and interrogated withnucleic acid probes having different optical labels associated withthem. By identifying the probes that hybridize, e.g., remain localized,within the confined area of the immobilized nucleic acid, one canidentify the sequence of the immobilized sequence. Likewise, where theimmobilized sequence is known, one can identify the sequence of theprobe sequences that hybridize to it.

In a further example, assays that detect differences in fluorescentpolarization capabilities of substrate and product may be monitoredusing the methods and systems of the invention. By way of example, U.S.Pat. No. 6,699,655, which is incorporated herein by reference in itsentirety for all purposes, describes homogeneous assay systems that arecapable of monitoring reactions in which reactants and products havesubstantially different charges. Such assays include kinase orphosphatase assays where phosphorylated or dephosphorylated productshave substantially different charges as compared to their substrates, asa result of addition or removal of a phosphate group, nucleic acidhybridization assays, protease assays, and the like. Briefly, a large,charged molecule or other structure associates differentially with asubstrate or product, based upon the charge differential, and thuschanges the rotational diffusion of the substrate or product,consequently changing the relative polarization of fluorescence emittedfrom an attached fluorescent label in response to polarized excitationradiation. In conjunction with the present invention, rather thandirecting the different planar components of depolarized fluorescence toseparate detectors, the two different signals are first spatiallyseparated, and then directed to different locations on the samedetector. An example of a system for use in performing applications thatdistinguish among different polarized optical signals is shown in FIG.4.

It will be appreciated that although described with respect to certaintypes of assays, the methods of the invention are useful in a variety ofdifferent analytical contexts where two or more optical signals emanatefrom a single confined source, but one desires to detect, record and/ormonitor them separately, including the use of internal control signals,and the like.

III. Systems

The present invention also provides for systems and devices useful incarrying out the above-described methods. FIG. 3 schematicallyillustrates one exemplary system for carrying out the methods of thepresent invention. As shown, the overall system 300 includes a source ofat least two different optical signals 302. As shown, source 302comprises a substrate that includes at least one, and preferably anarray of zero mode waveguides 304 fabricated thereon. An optical train306 is also provided that is in optical communication with the source302, including waveguides 304. As shown, optical train 306 includes asource of excitation radiation, e.g., a laser 308, laser diode, LED, orthe like, for use with fluorescent or fluorogenic optical signalingcomponents within the source 302. Also included in the optical trainshown 306, is a dichroic mirror 310 that reflects excitation radiationto direct it toward the source 302, e.g., including waveguide 304, butthat will pass emitted fluorescence. An objective lens or other focusinglens 312 is also typically provided to focus and further directexcitation radiation to and optical signals, e.g., fluorescence, fromsource 302. In the system illustrated, the signal is passed through abarrier or notch filter 314 to further reduce any excitation radiationnot reflected by dichroic 310, and then through a prism 316 or opticalgrating is provided to spatially separate excitation radiation by, e.g.,wavelength, and direct it through lens 312, and onto an opticaldetector, e.g., CCD 320. Useful prisms and/or optical gratings aregenerally commercially available from a variety of commercial opticssuppliers, including, e.g., Thorlabs, Inc. (New Jersey), Newport Corp(Irvine, Calif.), CVI Corporation (Alberquerque, N. Mex.), and the like.The signals detected upon CCD 320, including their intensity andlocation/pixel identification, are recorded by processor 322 which mayperform one or more data manipulations on such recorded signal data(e.g., to assign a reaction parameter, etc.) and then provided in a userfriendly readout format, e.g., on display 324.

Although shown as a single prism or grating, it will be appreciated thatin some cases, it may be desirable to use more than one prism. Inparticular, in some cases, the spatial separation of different signalsresulting from the dispersion profile of a given prism may not achieve adesired spatial separation. For example, in cases of high density ofdetector elements in a detector array, it may be desirable to providefor regularly or linearly spaced signal components. However, thedispersion profiles of given prism may not be linear, e.g., theresulting transmitted signals are not equally spatially separated.However, where detection is facilitated by ensuring all signals havesimilar separation relative to each other, e.g., in using CCDs fordetecting dense collections of signals, it may be advantageous tocombine prisms with dissimilar dispersion profiles to provide a nearlinear separation profile for each of the signals being detected.Likewise, in certain cases, detection of different signals may beoptimized by providing greater separation between two or more signalcomponents than a linear separation might afford. In such cases, thetunability of two or more prisms allows for this increased flexibilityof the system. In addition to the use of additional prisms or gratings,it will be appreciated that tuning of the system may be accomplished byrotating the prism or other dispersive optical element, e.g., around theoptical axis of the optical system and also perpendicular to thedirection of color separation, to adjust the degree of dispersion. Thus,in system embodiments, it may be useful to provide one or more of theprisms in a configuration that is capable of being readily rotated aboutthe axis.

In operation of the system shown, the source of different opticalsignals 302 includes a reaction mixture that generates products, orconsumes substrates that produce at least two different optical signals,e.g., substrates, intermediates and/or products that bear fluorescentlabels that emit light at differing wavelengths. Light source, e.g.,laser 308, directs excitation radiation, e.g., light at an appropriateexcitation wavelength for the fluorescent labels present in the source302, toward dichroic 310. The excitation radiation is reflected bydichroic 310, through objective 312, to impinge upon the source 302,thus exciting the fluorescent labels contained therein. The emittedfluorescence is again collected by objective 312 and directed throughdichroic 310, which is selected to reflect light of the wavelength ofthe excitation radiation, but pass light of the wavelength(s) of theemitted fluorescence. As a result, any reflected excitation radiation isfiltered away from the fluorescence. The fluorescent signal(s) are thendirected through a prism 316 or optical grating that spatially separatesthe differing signals by wavelength, and then refocused using a lens318, e.g., an imaging lens, and directs them to different locations onan optical detector array, e.g., CCD 320, photon counting avalanchephotodiode array, photomultiplier tube (PMT) array or the like. Avariety of different detector arrays may be employed in the invention,including, e.g., diode arrays, CCD arrays, and the like. CCDs aregenerally preferred for their compact nature, high resolution and cost,and may generally be employed as the detector. Various types of CCDs maybe employed to suit the needs of a given analysis, including, forexample, standard CCDs, electron multiplier CCDs (EMCCD), and/orIntensified CCD (ICCD).

As noted above, a modified system of the invention may be employed tomonitor signals that differ in other optical characteristics. Inparticular, FIG. 4 is a schematic illustration of a system that directsoptical signals that differ from each other in the relative polarity ofthe emitted fluorescence. Such detection may be employed in monitoringreactions that yield substantial size changes on products or reactants,and consequently changes in the reactant or product's ability to emitdepolarized fluorescence (See, e.g., U.S. Pat. No. 6,699,655). Bymeasuring light emitted in two orthogonal planes, one can assess therelative depolarization of fluorescent emissions in response topolarized excitation light. As shown, the system, 400, again includes anactivation light source 402 that is directed through a dichroic filter406 and objective 408 toward a confined reaction vessel or region 410.Light source 402 may comprise a polarized light source or be directedthrough a polarizing filter 404 to provide polarized excitationradiation to the reaction vessel 410. Emitted fluorescence is thencollected by objective lens 408 and directed through beam splitter 412,where it is split into two similar beams. Each beam is then separatelypassed through one of two oppositely polarized filters 414 and 416, suchthat only fluorescence in one of the two orthogonal planes is passedthrough lens 418 to each of the regions 422 and 424 on detector array420. The location of each signal on the detector array is an indicationof which plane of fluorescence is being detected. The intensity of thesignals are then compared to determine the relative depolarization offluorescence from the reaction mixture (See, again, U.S. Pat. No.6,699,655).

IV. Examples

To test the efficacy of the optical train in separating multiple opticalsignals from a confined source, a system was set up that wassubstantially similar to the system shown in FIG. 3. As shown, thesystem included a substrate having a series of zero-mode waveguidesfabricated thereon. The substrate was positioned proximal to and withinoptical communication of objective lens, and a white light source waspositioned above the zero mode waveguide substrate and directed througha narrow band filter, at the waveguide substrate. An objective lens wasused to focus optical signals from the waveguides through wedge prism.Once separated by wedge prism, the different optical signals were thenpassed through the imaging lens onto a 512×512 pixel EMCCD camera chip.In operation, the broadband light (made up of a subset continuum of thewhite light spectrum), collected by the objective lens and then passedthrough a wedge prism was then focused, as a collection of separatedsignals, upon the CCD camera. FIG. 5A illustrates the images derivedfrom four different regions of the CCD, corresponding to light from theeight different zero mode waveguides and four different wavelengths, 405nm (A), 488 nm (B), 568 nm (C) and 647 nm (D). FIG. 5B is a plot of therelative location, in distance from a position of an unseparated signal,in microns, showing the relative separation distance between theseparated signals.

A comparison experiment was also performed to demonstrate the increasedefficiency of the prism based separation as compared to a filter basedwavelength separation. In particular a mixture of two differentfluorescent dyes (Alexa488 and Alexa568, available from MolecularProbes, Eugene, Oreg.) having different peak emission wavelengths (488nm and 568 nm, respectively) was prepared and interrogated usingappropriate excitation radiation. Emissions from the mixture were passedthrough an objective and subjected to either filter based wavelengthseparation (using two Semrock triple notch filters, or wedge prism basedseparation, prior to focusing the separated signals onto a CCD chip. Thetable, below, provides fluorescence intensities of each signal in eachdifferent optical train, as measured using an EMCCD. As can be seen, theprism based separation yields substantially higher efficiency detectionof the separated signal as compared to the filter based system.

Fluorescent Intensity Detected Separation Method Alexa488 Alexa568Filter based separation 1146 1263 Prism Separation 2845 2676

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. Unless otherwise clear from the context or expresslystated, any concentration values provided herein are generally given interms of admixture values or percentages without regard to anyconversion that occurs upon or following addition of the particularcomponent of the mixture. To the extent not already expresslyincorporated herein, all published references and patent documentsreferred to in this disclosure are incorporated herein by reference intheir entirety for all purposes.

1-40. (canceled)
 41. A method of identifying nucleotides in a nucleicacid sequence, comprising: providing a plurality of polymerizationcomplexes within a plurality of confined reaction environments, whereineach complex comprises a polymerase enzyme, and a template nucleic acid;contacting the plurality of complexes with a plurality of types ofnucleotide analogs labeled with distinguishable fluorescent labels underconditions suitable for polymerization; transmitting fluorescent signalsassociated with incorporation of a nucleotide analog to a detector,wherein location of the fluorescent signals on the detector isindicative of an individual confined reaction environment and a type ofnucleotide incorporated; identifying a nucleotide in a nucleic acidsequence based upon the type of nucleotide incorporated and the confinedreaction environment.
 42. The method of claim 41, wherein thetransmitting step comprises collecting fluorescent signals from theplurality of confined reaction environments and passing the fluorescentsignals through an optical train that directs different spectralcomponents of the fluorescent signals to different locations on thedetector.
 43. The method of claim 42, wherein the optical traincomprises a dispersive optical element selected from a prism and anoptical grating.
 44. The method of claim 41, wherein the confinedreaction environments comprise zero mode waveguides.
 45. The method ofclaim 41, wherein fluorescent signals associated with incorporation of aplurality of nucleotide analogs in a complex are transmitted to thedetector, and a plurality of nucleotides in the nucleic acid sequenceare identified.
 46. The method of claim 41, wherein the optical traincomprises at least a first prism to direct spectrally differentfluorescent signals to different locations on the detector.
 47. Themethod of claim 46, further comprising at least a second prism,configured to adjust relative spectral separation for differentfluorescent signal components.
 48. The method of claim 47, wherein thesecond prism is provided to be rotatable on its optical axis.
 49. Amethod of identifying nucleotides incorporated in polymerase mediated,template dependent nucleic acid synthesis reactions comprising:providing a plurality template nucleic acid/polymerase complexes on asubstrate; contacting the plurality of complexes with a plurality ofnucleotide analogs having spectrally distinct fluorescent labels,wherein incorporation of nucleotides produces characteristicincorporation signals; directing incorporation signals to a singledetector, wherein a location of a signal on the detector is indicativeof a complex incorporating the nucleotide and a type of nucleotideincorporated; and identifying the nucleotide incorporated into atemplate mediated nucleic acid synthesis reaction from the location ofthe signal on the detector.
 50. The method of claim 49, wherein thedirecting step comprises passing optical signals through dispersiveoptical element to separate signals from the spectrally distinct labelsand image the signals onto different regions of the single detector. 51.The method of claim 49, wherein the complexes are individually opticallyresolvable.
 52. The method of claim 49, wherein the plurality ofcomplexes are optically confined.
 53. The method of claim 52, whereinthe plurality of complexes are disposed within an array of zero modewaveguides.
 54. A method of analyzing a reaction, comprising: providinga first reactant immobilized on a substrate; contacting the firstreactant with a second reactant and a third reactant, each bearing aspectrally distinct label; directing signals from the spectrallydistinct labels that are characteristic of interaction of the firstreactant with one of the second and third reactants to a location on afirst detector, wherein the location on the detector is indicative ofthe first reactant location on the substrate and the second or thirdreactant with which the first reactant interacted; identifying the firstreactant and the second or third reactant with which the first reactantinteracted.
 55. The method of claim 54, wherein the first reactantcomprises at least a first nucleic acid.
 56. The method of claim 55,wherein the first reactant further comprises a polymerase enzyme. 57.The method of claim 55, wherein the second and third reactants comprisesecond and third nucleic acids, respectively.
 58. The method of claim56, wherein the second and third reactants comprise first and secondnucleotide analogs, respectively
 59. The method of claim 57, wherein thesecond and third nucleic acids each comprise a spectrally distinctfluorescent label.
 60. The method of claim 58, wherein the first andsecond nucleotide analogs each comprises a spectrally distinctfluorescent label.